Determination of serum triglyceride levels in mammalian blood is of importance because elevated levels may be useful in the diagnosis of certain maladies. For example, determination of serum triglycerides is used to screen for coronary artery diseases, diabetes mellitus, nephrosis, biliary obstruction and various metabolic disorders caused by endocrine disturbances.
Triglycerides have the general structural formula shown in the following equation: ##STR1## Determination of triglyceride levels is carried out by splitting the triglyceride as indicated above, to liberate free fatty acids (FFA) and glycerol.
Early procedures for liberation of glycerol and FFA involved saponification by addition of an alcoholic solution of alkali metal hydroxide. The procedures required arduous and time-consuming steps, such as the use of elevated temperatures, removal of materials such as phospholipids, glucose and bilirubin, deproteinization and neutralization with acid.
The number of triglyceride determinations performed in clinical laboratories has steadily increased in the past three years, producing a demand for a rapid method of liberation of glycerol and determination of triglycerides. Therefore, totally enzymatic methods for triglyceride determination have rapidly gained in favor. These methods involve the sequence of enzymatic hydrolysis of triglycerides followed by enzymatic determination of the glycerol liberated. For a review of the principles involved, see Clin. Chem. 19, 476 (1973) and 21, 1627 (1975).
One of the first approaches used in the enzymatic hydrolysis of triglycerides involved the use of animal source enzymes, e.g., pancreatic lipase, isolated from beef pancreas. The enzyme was unsatisfactory because of the incomplete hydrolysis of the triglycerides into glycerol; incomplete hydrolysis results in a mixture of mono and diglycerides. The use of microbial source lipase was then investigated. Again, because of problems with incomplete hydrolysis, unsatisfactory results were obtained. Consequently, research to improve enzymatic hydrolysis was undertaken by numerous laboratories. This research interest is reflected in the patents referred to below.
U.S. Pat. No. 3,703,591 discloses an enzymatic method for hydrolysis of triglycerides which involves adding to the triglycerides a mixture of lipase and a protease. The presence of protease causes a reduction in the stability of the enzyme test system.
U.S. Pat. No. 3,898,130 discloses an enzymatic method for hydrolysis of triglycerides which involves the use of a combination of a lipase and a bile salt, such as sodium taurodeoxycholate.
U.S. Pat. No. 4,056,442 discloses an enzymatic method for hydrolysis of triglycerides which involves the use of two microbial lipases, obtained from Rhizopus arrhizus and Candida cyclindracea, to overcome some of the disadvantages of the above-described lipase-containing mixtures. The amount of lipase required is a significant disadvantage; approximately 70 units of R. arrhizus and 64 units of C. cylindracea are required for complete hydrolysis.
U.S. Pat. No. 3,862,009 discloses an enzymatic method for hydrolysis of triglycerides which involves the use of a lipase from R. arrhizus, carboxyl esterase and an alkali metal or alkaline earth metal alkyl sulfate of 10-15 carbon atoms. The carboxyl esterase is specific for the hydrolysis of lower fatty acid esters, such as methyl or ethyl butyrate, whereas cholesterol esterase is specific for the hydrolysis of steroid esters.
U.S. Pat. No. 4,066,508 discloses an enzymatic method for hydrolysis of triglycerides which involves adding a polyglycol to the composition claimed in the '009 patent.
None of the patents discussed above disclose or suggest an economical composition and rapid method for triglyceride determination by use of an enzymatic composition of lipase and cholesterol esterase.